Simultaneous technological and conceptual advances in molecular microbiology, i.e., the polymerase chain reaction (PCR) and molecular phylogenetics, together form a powerful new experimental approach that will be used to investigate the nature of a recently identified, but uncultivated bacterial pathogen, and the etiology of a common but idiopathic chronic inflammatory disease. Little is known concerning the novel bacterium Tropheryma whippelii, except its putative role as the causative agent of Whipple's disease and its phylogenetic relationships, as revealed by small subunit or 16S ribosomal DNA (rDNA) sequence analysis. The proposed research will help to elucidate the causative role, natural ecology, detailed phylogeny, and physiological characteristics of the Whipple bacillus, as well as the spectrum of disease with which it is associated. This information will improve our understanding of disease pathogenesis and prevention, as well as expand our understanding of the broad family of medically-useful actinomycetes. Although the etiology of sarcoidosis is unknown, an infectious etiology has been suspected for this chronic inflammatory disease. The epidemiology of sarcoidosis, the known limitations of laboratory culture, and the similarities between sarcoidosis and other known infectious diseases (such as Whipple's Disease), suggest that an uncultured pathogen(s) may be the cause of sarcoidosis. A molecular approach will be used to search for an uncultured pathogen in sarcoidosis, based on the successful model previously used in this laboratory to identify the Whipple bacillus. This approach is based upon broad range (bacterial and fungal) PCR amplification of small subunit rDNA sequences directly from diseased human tissue. RESEARCH QUESTIONS: 1. What can we learn about the physiology, basic biology, and biologically active by-products of the Whipple bacillus? A search will be made for homologues of genes that are commonly found in other actinomycetes, such as those encoding biodegradative enzymes and antibiotics, and other genes with phylogenetically useful information, by using consensus PCR and gene probes. (Phase I) 2. Can the argument for a causative role of T. whippelii in Whipple's disease be strengthened by means of a specific T. whippelii 16S rRNA probe and tissue in situ hybridization? Confocal laser fluorescence microscopy will be used to try to co-localize bound probe and bacteria within Whipple's disease tissues, including samples from the 1907 original case. (Phase I) 3. What is the spectrum of disease associated with the Whipple bacillus, and where is the bacterium found in nature? Whipple specific PCR primers will be employed to search for this bacillus in tissues from patients with AIDS-associated chronic diarrhea, and chronic uveitis, arthritis and meningitis. The same assay will be applied to stool samples from healthy humans and animals, soil and environmental water samples. (Phase II) 4. Is sarcoidosis an infectious disorder? Broad range bacterial and fungal ss rDNA PCR primers will be used to target phylogenetically informative sequences from sarcoidosis tissues and Kveim spleen. In particular, is the Whipple bacillus one cause of this syndrome? (Phase II)